Method of recovering a nucleic acid encoding a proteinaceous binding domain which binds a target material

Abstract

In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

We claim: 
     
       1. A method of recovering a nucleic acid encoding a proteinaceous binding domain, the method comprising:
 providing a variegated population of filamentous phage, wherein each phage, displays on its outer surface a proteinaceous potential binding domain that is physically associated with a nucleic acid sequence that encodes the potential binding domain, the potential binding domains differing through the at least partially random variegation of one or more amino acid positions of a parental binding domain; 
 contacting the phage with a target material such that the potential binding domain and the nucleic acid that encodes the potential binding domain remain physically associated, and such that the potential binding domain and the target material may interact; 
 isolating at least one binding domain that binds to the target material; and 
 recovering the nucleic acid that is physically associated with the at least one isolated binding domain during the contacting, 
 wherein the parental binding domain comprises an antibody domain, and at least one of said variegated amino acid positions is within a hypervariable region of the antibody domain. 
 
     
     
       2. The method of  claim 1  wherein the variegation excludes cysteines. 
     
     
       3. The method of  claim 1  wherein the parental binding domain is a domain of a naturally occurring protein. 
     
     
       4. The method of  claim 1  wherein the parental binding domain is a non-naturally occurring domain which substantially corresponds in sequence to a naturally occurring domain. 
     
     
       5. The method of  claim 4  wherein the parental binding domain differs from the corresponding naturally occurring domain in sequence by one or more substitutions, insertions, or deletions. 
     
     
       6. The method of  claim 1  wherein the parental binding domain substantially corresponds in sequence to a hybrid of subsequences of two or more naturally occurring proteins. 
     
     
       7. The method of  claim 1  wherein the recovering comprises removing the at least one isolated binding domain from the filamentous phage that physically associates the at least one isolated binding domain with the particular nucleic acid sequence that encodes it during the contacting. 
     
     
       8. A method of recovering a nucleic acid encoding a binding domain, the method comprising:
 providing a variegated population of filamentous phage, wherein each phage displays on its outer surface a potential binding domain that comprises an antibody domain and is physically associated with a nucleic acid sequence that encodes the potential binding domain, the encoded potential binding domains differ from one another through the at least partially random variation of one or more amino acids corresponding to a hypervariable region, and the random variation of at least one of the amino acid positions is by random selection of the codon encoding the amino acid at said position from a set of codons, the set being characterized by one or more of the following properties: 
 (a) the set includes at least one codon for each of at least two different amino acids other than cysteine, and excludes all codons encoding cysteine, 
 (b) the set provides a single codon for each encoded amino acid, and 
 (c) the amino acids encoded by the set are represented at substantially equal frequency; 
 contacting the phage with a target material such that the potential binding domains and the target material may interact while each potential binding domain and a nucleic acid sequence that encodes it remain physically associated; 
 isolating a binding domain that binds to the target material; and 
 recovering the particular nucleic acid that is physically associated with the isolated binding domain during the contacting. 
 
     
     
       9. The method of  claim 8  wherein the set excludes all codons encoding cysteine. 
     
     
       10. The method of  claim 8  wherein the set provides a single codon for each encoded amino acid. 
     
     
       11. The method of  claim 8  wherein the amino acids encoded by the set are represented at substantially equal frequency. 
     
     
       12. The method of  claim 8  wherein the set is characterized by all three properties, (a), (b), and (c). 
     
     
       13. A process for determining a binding property of a proteinaceous domain, the process comprising:
 mutagenizing a gene encoding an antibody domain to form a gene encoding a potential binding domain, wherein at least one codon encoding an amino acid in a hypervariable region is mutagenized; 
 displaying the potential binding domain on the outer surface of an amplifiable genetic package, wherein said amplifiable genetic package is a filamentous bacteriophage that contains the gene encoding said potential binding domain, 
 contacting the package with the target material, and 
 determining whether the package displaying the potential binding domain binds to said target material. 
 
     
     
       14. The process of  claim 13  wherein the at least one codon is a codon selected from a set of codons, the set excluding all codons encoding cysteine. 
     
     
       15. A method of isolating a binding protein, the method comprising:
 preparing a variegated population of filamentous phage, each phage including a nucleic acid construct that encodes a chimeric protein that comprises a potential binding domain and a polypeptide encoded by a filamentous phage gene VIII or a filamentous phage gene III, the encoded potential binding domains differ through the at least partially random variation of one or more amino acid positions of a parental binding domain; 
 expressing the potential binding domains encoded by the nucleic acid constructs of the phage population; 
 isolating at least one phage from the population, the isolated phage including a nucleic acid construct that encodes a potential binding domain that binds to the target material with at least a predetermined affinity; and 
 recovering the nucleic acid construct from the isolated phage. 
 
     
     
       16. The method of  claim 15  wherein the chimeric protein comprises a potential binding domain and a mature filamentous phage gene VIII protein. 
     
     
       17. The method of  claim 15  wherein the chimeric protein comprises a potential binding domain and a mature filamentous phage gene III protein. 
     
     
       18. The method of  claim 1 , wherein the outer surface protein is a coat protein. 
     
     
       19. The method of  claim 1 , wherein the outer surface protein and the potential binding domain are assembled periplasmically. 
     
     
       20. The method of  claim 18 , wherein the coat protein is pIII. 
     
     
       21. The method of  claim 18 , wherein the coat protein is pVIII. 
     
     
       22. The method of  claim 1 , wherein the potential binding domain and the outer surface protein are displayed as a chimeric protein on the phage surface. 
     
     
       23. The method of  claim 1 , wherein the nucleic acid is contained within a phagemid. 
     
     
       24. The method of  claim 1 , wherein recovering comprises eluting the phage from the target material. 
     
     
       25. The method of  claim 1 , wherein recovering comprises cleaving the phage from the target material. 
     
     
       26. The method of  claim 25 , wherein the cleavage involves cleaving the binding domain from the phage.